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Robert D. Combes

The work of Dr Richard Clothier (Reader in Cellular Toxicology and Director of the FRAME Alternatives Laboratory in the University of Nottingham Medical School) in relation to the FRAME Research Programme, is reviewed. He made a very substantial contribution to FRAME’s laboratory research work over the last 20 years, by publishing many research papers, mainly with respect to method development and the application of new replacement toxicity test methods, particularly those involving the use of human cells in tissue culture. In addition, he participated in a number of international validation studies that have facilitated the regulatory acceptance of certain new methods. Lastly, he has been closely involved in the logistical aspects of obtaining and maintaining external research funding for FRAME from industrial sponsors.

ATLA Staff Writer

A one-day symposium was held at the University of Nottingham Medical School, Queen’s Medical Centre (QMC), on 30 November 2005, in recognition of the retirement of Dr Richard Clothier from the School of Biomedical Sciences (SBS).

Michael Balls

The career of Richard Clothier is reviewed in the light of his long-standing collaboration with Michael Balls and Laurens Ruben at the University of East Anglia (UEA), the University of Nottingham, and Reed College, Portland, Oregon, USA. It began with work at UEA on the aetiology of the lymphosarcoma of Xenopus laevis, followed by studies on the effects of exposure to N-nitroso-N-methylurea on T-cell functions, which led to many contributions to comparative immunology. This was followed by the establishment of the FRAME Research Programme, which led to participation in extensive studies on the development of in vitro cytotoxicity tests and their application in acute and topical toxicity testing. A FRAME Trustee since 1983, Richard Clothier was a co-founder, and subsequently Director, of the FRAME Alternatives Laboratory in the University of Nottingham Medical School, where he led successful collaborations with a number of industrial partners and, in particular, with the European Centre for the Validation of Alternative Methods (ECVAM).

Current approaches to predicting adverse effects in humans from acute toxic exposure to cosmetic ingredients still heavily necessitate the use of animals under EU legislation, particularly in the context of the REACH system, when cosmetic ingredients are also destined for use in other industries. These include the LD50 test, the Up-and-Down Procedure and the Fixed Dose Procedure, which are regarded as having notable scientific deficiencies and low transferability to humans. By expanding on previous in vitro tests, such as the animal cell-based 3T3 Neutral Red Uptake (NRU) assay, this project aims to develop a truly animal-free predictive test for the acute toxicity of cosmetic ingredients in humans, by using human-derived cells and a prediction model that does not rely on animal data. The project, funded by Innovate UK, will incorporate the NRU assay with human dermal fibroblasts in animal product-free culture, to generate an in vitro protocol that can be validated as an accepted replacement for the currently available in vivo tests. To date, the project has successfully completed an assessment of the robustness and reproducibility of the method, by using sodium lauryl sulphate (SLS) as a positive control, and displaying analogous results to those of the original studies with mouse 3T3 cells. Currently, the testing of five known ingredients from key groups (a surfactant, a preservative, a fragrance, a colour and an emulsifier) is under way. The testing consists of initial range-finding runs followed by three valid runs of a main experiment with the appropriate concentration ranges, to generate IC50 values. Expanded blind trials of 20 ingredients will follow. Early results indicate that this human cell-based test holds the potential to replace aspects of in vivo animal acute toxicity testing, particularly with reference to cosmetic ingredients.

Mark J. Andrews, Michael J. Garle and Richard H. Clothier

Alamar BlueTM (AB) is a new tetrazolium dye substrate that has been introduced as an alternative cell viability indicator. AB is reduced by intracellular reductases to a product which is exported from cells and can be quantified by fluorescent or spectrophotometric methods. We investigated the processes by which AB was reduced in liver cytosolic, microsomal or mitochondrial fractions and in cultured rat hepatocytes. AB reduction was catalysed by all liver fractions in an NADPH-dependent and NADH-dependent manner; the cytosolic fraction catalysed the highest rate of AB reduction. All of these activities were inhibited by dicoumarol (10μM), except AB reduction catalysed by NADH in mitochondrial fractions, which was resistant to the effects of dicoumarol and the metabolic inhibitors, but sensitive to inhibition by mercury (II) chloride. In hepatocyte cultures, AB reduction was stimulated by dicoumarol (10μM), menadione (10μM), rotenone (10μM), lactate (1–10mM) and fluoride (3–10mM). Potassium cyanide, ethanol and malonate had little effect. The results from this study suggest that AB is reduced in an NADPH-quinone oxidoreductase-dependent fashion, but that superoxide may also be involved in the reduction of AB. The modulation of AB reduction by lactate means that AB reduction may be modified by alterations in intermediary metabolism which are not a reflection of cell lethality. Therefore, great care should be exercised when using AB reduction as a viability indicator.

The Alamar BlueTM reduction assay is used as an indicator of cellular viability in in vitro tests for the prediction of ocular irritancy. Alamar Blue itself has a low cytotoxicity, so repeat exposure and recovery studies can be performed on the same cells. This paper contains the results of a preliminary investigation of interactions between the Alamar Blue dye and a confluent monolayer of epithelial Madin-Darby canine kidney cells. This was performed by using an experimental fluorescence microscope and differential phase confocal microscope designed for studying live samples in vitro. The initiation of Alamar Blue reduction to its fluorescent product did not occur at the same time in all cells, but started in a small number and spread progressively through their immediate neighbours. There was clear localisation of the reduced (fluorescent) Alamar Blue within the nuclei and other organelles.